An electrochemical analysis method can be used to both qualitatively and quantitatively define substances on the basis of specific physical characteristics, using electric current. Electrochemical analysis methods in which electrode reactions play a role are of particular importance. Depending on whether the excitation signal (current, voltage or potential) is kept constant, these methods are subdivided into two groups. For example, potentiometry, chronopotentiometry, coulometry, amperometry, chronoamperometry and chronocoulometry are techniques in which the excitation signal is kept constant. In voltammetric and polarographic methods, the excitation signal is varied.
Together with optical methods, electrochemical analysis methods such as these for analytical definition of chemical and biochemical substances are characterized by high sensitivity as well as high selectivity. However, while complex, expensive and sensitive optical and optoelectronic appliances are required for optical analysis methods, electrochemical analysis methods require only comparatively simple electrode apparatuses. One major advantage of electrochemical analysis methods is the direct presence of the measurement result in the form of an electrical signal. This can be processed further, after analog/digital conversion, directly by a computer, preferably by a personal computer.
Electrochemical analysis methods are suitable for qualitative and quantitative measurement of substance concentrations in an electrolyte solution. Each substance has an oxidation voltage or reduction voltage which is characteristic of it. These voltages can be used to distinguish between different substances. Furthermore, the concentration of the substance under consideration can be deduced from the electric current which flows during a reaction.
An electrochemical experiment requires at least two electrodes which are connected (working electrode, opposing electrode) to the substance (electrolyte) to be analyzed. However, a plurality of working electrodes can also be used in parallel. A reference electrode is generally also used in order to exactly monitor the electrolyte potential. This system, which has at least three electrodes, is connected to a potentiostat, which allows regulation of the potential at the working electrode and measures the electric current flowing through the working electrode.
In the case of voltammetry, a variable voltage is applied to the working electrode and the current flowing during oxidation or reduction is measured. In the specific case of cyclic voltammetry, a specific voltage range is covered repeatedly in such a manner that the substances contained in the electrolyte are oxidized and reduced a plurality of times successively.
In the case of chronoamperometry, a defined voltage is applied suddenly to the working electrode, and the current flowing is recorded over time. This measurement method allows the analysis of one specific substance by deliberate oxidation or reduction of this substance. The current flowing is a measure of the amount of substance converted per unit time, and allows conclusions to be drawn about the concentration of the substance and of the diffusion constants.
Chronocoulometry corresponds to chronoamperometry, in terms of the electrical constraints. However, in contrast to this, the total amount of electrical charge that has flowed is recorded rather than the electric current flowing.
In the refinement as sensors, electrode apparatuses can be used in different electrochemical analysis methods. The only critical factor is that substances which can be evaluated electrochemically are produced when a sensor event occurs. For example, a marking method which produces electrochemical substances when a sensor event occurs is used for sensors for detection of biomolecules.
Miniaturized electrochemical electrode systems for analysis of chemical and biochemical substances are known in the prior art [1], [2], [3], [4] and [5]. The electrodes of arrays such as these can be made contact with individually at the edge of the substrate, and can be operated by means of a potentiostat. In order additionally to provide electrode arrays which, for example, have 100 or more electrodes, switching functions on the substrate are advantageous, which multiplex the electrodes onto common connecting lines. If the substrate is a semiconductor material such as silicon, the switches required can be provided by MOS transistors, as described in [6]. Since the tests can in this case be carried out in parallel, the analysis time is considerably shortened, and it is also possible to carry out complex analyses.
Reference [7] describes the so-called EDDA method (Electrically Detected Displacement Assay Method), from the Friz BIOCHEM™ Company.
From the point of view of miniaturization, signal integrity and measurement sensitivity, the active micro arrays which are known from the prior art represent very good electrochemical analysis systems [2]. In this case, not only the multiplexing and/or selection functions but also the amplification, the conditioning of the signals and, possibly, also the evaluation of the signals are integrated in the semiconductor material. These sensor arrays are referred to as active arrays since, in contrast to passive arrays, active electronics process signals on the chip. Active electrochemical sensor arrays such as these, which operate using voltammetric (chrono)amperometric and (chrono)coulometric methods are manufactured using CMOS technology and are equipped with electrodes which are accessible on the chip surface and are composed of a noble metal (for example gold).
By way of example, active sensor arrays have been implemented for DNA sensor chips, in which redox cycling is used as the basis for verification of DNA molecules on surfaces electronically by detection of electrical charge carriers that are produced by means of redox-active substances. Redox cycling represents a special case of an amperometric method (oxidation/reduction voltages constant, measurement of the electrode current).
A typical redox cycling sensor arrangement has two gold electrodes formed on a substrate. By way of example, immobilized single-strand DNA capture molecules with a predetermined sequence are immobilized on each electrode by means of so-called gold-silver coupling. The complementary single-strand DNA target molecules which may be present in the analyte solution and thus have the capability for hybridization have a marking.
When suitable additional molecules are present, this marking is used to initiate a cycle comprising oxidation and reduction of components of the additional molecules, leading to the formation of reduced or oxidized molecules by interaction with the electrodes. The cycle comprising oxidation and reduction processes leads to an electrical circulating current which allows verification of the DNA target molecules.
The opposing electrode is always required both for this redox cycling sensor arrangement and for the already mentioned electrochemical analysis methods. However, while only a relatively small direct current need be dissipated to the electrodes in the case of redox cycling sensors, a comparatively high surge current must be able to be supplied by the opposing electrode in most of the electrochemical analysis methods mentioned above. For this reason, the area of the opposing electrode must be considerably larger than that of the active working electrodes.
Depending on the specific analysis method, the opposing electrode generally needs to have a surface area which is about 10 times larger than the sum of the surface areas of the individual working electrodes. This is necessary because, if the area of the opposing electrode is too small, the voltage which is applied to it can assume extremely high values in order to produce the charge carriers that are required for an experiment. When high values such as these are assumed, this can result in chemical reactions taking place in an uncontrolled manner, for example with the electrode material, and these typically lead to the formation of gases.
If the surface area of the opposing electrode is large enough, it is able to stabilize the electrolyte potential most of the time, by way of the double-layer capacitance. Electrochemical reactions take place only with comparatively low current densities.
Since an active silicon chip is comparatively expensive as a substrate, for example for a DNA sensor, it is generally desirable for the individual sensors in the array to be packed as densely as possible. In some circumstances, the packing density of the sensors and thus of the electrodes in the area of the sensor array can mean that it is not possible to provide an opposing electrode. The opposing electrode can then be in the form of an external electrode, which is arranged in the sample volume and is electrically connected to the sensor chip. This electrode can be driven from a potentiostat.
However, this procedure is disadvantageous because of the comparatively long supply lines and the more complex mechanical design. If the disadvantages associated with this are intended to be avoided, the only solution available from the prior art is for the opposing electrode to be formed in the periphery of the array, but this results in additional (expensive) chip area being required.
Reference [8] describes an electrode system for detection of molecules or molecule complexes. The arrangement according to [8] contains three electrodes, specifically a working electrode, an opposing electrode and a reference electrode. The reference electrode is arranged in such a manner that it is adjacent to at least subareas of the two further electrodes. Evaluation circuits are integrated in the substrate, with the working electrode and the electrode pair comprising the opposing electrode and the reference electrode being firmly connected to evaluation circuits, which are separate from one another.
Reference [9] describes a circuit for switching between different electrodes in an electrolysis apparatus. The electrolysis apparatus has an auxiliary electrode, a reference electrode, two working electrodes and a separation electrode. In a first working phase, a first working electrode is connected to a potentiostat, while the second working electrode is connected to the separation electrode. An electrolyte is deposited in a reference solution on the separation electrode. In a second working phase, the electrolyte which has been deposited on the separation electrode is separated from it, and a current which occurs during this process is measured.
Reference [10] describes a DNA sensor having interdigital electrodes. The interdigital electrodes have additional reaction surfaces for the accumulation of thiols.
Furthermore, [11] describes a biosensor having three electrodes, with the first electrode having a holding area for holding capture molecules. The second electrode and the third electrode are designed in such a manner that a redox process takes place on them in the course of a redox cycling process.
Furthermore, [12] describes a biosensor having a unit for immobilization of biopolymers. Furthermore, a detection unit is provided for detection of biopolymers, which are bonded to capture molecules which are applied to the unit for the immobilization of biopolymers, as well as a thermostatization unit which is designed to separate complexes comprising capture molecules and detected biopolymers by raising the temperature to a temperature above the melting point of the complexes.
References [13] and [14] describe methods for detection of biopolymers, in which capture molecules which have not been hybridized are removed from biopolymers with capture molecules using a hybridization process, and the hybridized biopolymers are detected after the removal process.